Severe acute respiratory syndrome is a life-threatening respiratory disease that probably originated in Guangdong Province, China in the fall of 2002 [1, 2]. The agent responsible for the disease spread rapidly [3,4]. A novel coronavirus (SARS-CoV), isolated from febrile and dying patients, is the etiologic agent responsible for the disease [5-8]. SARS-CoV infection is associated with overall case fatality rates thought to approach ˜14-15%, with selected populations being at increased risk (>50% in the elderly). SARS-CoV has infected over 8,000 individuals worldwide and caused over 800 deaths, before aggressive infection control measures successfully contained the scope of the outbreak. Despite intensive efforts, no effective antiviral treatments against SARS have been described.
Coronaviruses, members of the order Nidovirus, contain the largest single-stranded, positive-polarity RNA genome in nature and are divided into three main serogroups; group I: transmissible gastroenteritis virus (TGEV) and human coronavirus 229E (HCV-229E), group II: mouse hepatitis virus (MHV) and bovine coronavirus (BoCV), and group III: infectious bronchitis virus (IBV). Sequence analyses suggest that SARS-CoV represents the prototype strain of group IV [6, 8-10]. The SARS-CoV genomic RNA is ˜29,700 base pairs in length and has several large open reading frames (ORFs) encoded in subgenomic and full-length mRNAs [8-10]. The subgenomic mRNAs are arranged in the form of a nested set from the 3 proximal end, and leader RNA sequences, encoded at the 5′ end of the genome, are joined to body sequences at a highly conserved consensus sequence (CS) located just upstream of each of the ORFs. The exact SARS CS sequence has been reported as either CUAAAC or AAACGAAC by different laboratories [8,9]. The SARS-CoV genome length RNA is likely packaged by a 50-kDa-nucleocapsid protein (N) [8]. As with other coronaviruses, the virion contains several viral structural proteins including the ˜140 kDa spike glycoprotein (S), a 23 kDa membrane glycoprotein (M) and a ˜10 kDa protein (E).
The coronavirus gene 1, or replicase gene, comprises two-thirds of the genome. MHV contains two overlapping open reading frames, ORF1a and ORF1b, which are connected by a ribosomal frameshift structure. In MHV, three proteinases, papain-like proteinases 1 and 2 (PLP-1, PLP-2) [11-13] and 3C-like proteinase (3CLpro) [14], are expressed as part of the replicase gene polyprotein and mediate cleavage of the polyproteins into at least 15 mature proteins. Continuous protein processing is crucial for ongoing virus transcription so MHV replication is sensitive to protease inhibitors that prevent replicase processing [11]. Additional functions have been predicted for proteins processed from the replicase polyprotein, including an RNA-dependent RNA polymerase (pol), an RNA helicase (hel) and a capping enzymatic activity [6, 15, 16]. The SARS virus replicase gene is similarly organized except that the SARS virus replicase has been predicted to encode only the PLP-2 equivalent and the 3CLpro proteases [6, 7].
The present invention provides a full length cDNA of the SARS coronavirus, from which transcripts are produced that replicate and/or are infectious in vitro or in vivo, multiplication-defective replicon vector particles produced from the cDNA and methods of making and using these compositions as immunogens, vaccines and/or nucleic acid delivery vectors.